JOURNAL OF COSMETIC SCIENCE 240 muscle cell innervation using spinal cord and spinal ganglion explants from rat embryos. The cells were treated with lyophilized extract of Acmella oleracea diluted to 50% in maltodextrin or pure sp ilanthol (40 × 10-5% or 160 × 10-5%). The frequency of contrac- tion in nerve–muscle coculture with spilanthol was determined after 5 min, 1 h, and 6 h of incubation with the substance. Both extract and pure spilanthol, applied externally, reduced muscle tension, thus hopefully contributing to the reduction of facial expression wrinkles (65). Spilanthol perfectly overcome s the epidermal barrier and migrates deep into the skin. Veryser et al. (66) analyzed the transdermal behavior of spilanthol using human skin in a Franz diffusion cell setup. The dose solutions (30% w/w spilanthol in ethanol or products with spilanthol) were applied on the 0.64 cm2 skin surface with a micropipette. Authors noted that spilanthol penetrates through the human stratum corneum and the viable epidermis, thereby reaching the dermis and thus the systemic circulation, with permea- bility coeffi cient (Kp) values between 0.6 and 53.3 × 10–4 cm/h, depending on i.a. the used dose formulation. Cosmetics based on spilanthol as an active ingredient are gaining popularity, especially anti-wrinkle products (mainly creams). They also occur in the form of water extracts from Acmella oleracea, used to wash the skin. Cosmetics containing spilanthol are also gels, care emulsions in types oil/water or water/oil, or nanoemulsions (67). They are mainly similar to botu linum toxin activity. It is responsible for the reduction or complete elimination of unaesthetic skin creases in various areas of the face—from very fi ne to deep wrinkles. The effects of cosmetic products containing spilanthol are compa- rable to those after using botulinum toxin. Hence, its name is “herbal botox.” Spilanthol is safe and nontoxic. No contraindications for its use have been shown. Spilanthol proper- ties were studi ed on nerve–muscle coculture model, which is a model suited to studying the infl uence of a substance on muscle contraction frequency, as well as to studying the recuperation of contractile activity after blockage of muscle contractions by a substance. The frequency of contraction was determined after 5 min, 1 h, and 6 h of incubation with spilanthol. At 6 h, the substance was eliminated, and recuperation of contractile activity was studied 1 h and 24 h later. At the concentrations 40 × 10-5% and 160 × 10-5%, pure spilanthol blocked muscle contractions after 5 min of incubation. The blockage was maintained until 6 h and the fi bers remained blocked for 24 h after elimination of the substance (65). Spilanthol works in a variety of ways , including larvicidal (10–14 μg/mL), antimicro- bial (25–300 μg/mL), and fungicidal and bacteriostatic (5–150 μg/mL). This com- pound also has anti-infl ammatory properties (90–180 μM in the macrophage cell line), and antianxiety (3–30 mg/kg i.p. in mice) and diuretic (800 mg/kg per os in mice) ef- fects (68,69). To estimate the bacteriostatic activity o f spilanthol, Molina Torres et al. (70) used liquid cultures, which were grown at 28 or 37°C, depending on the microorganism, in the PDB medium with vigorous agitation. The growth of E. coli (Gram -) and B. subtilis (Gram +) was 100% inhibited at spilanthol concentrations of 75 and 150 μg/mL, respectively. E. carotovora (Gram -) was not sensitive to the higher concentration of this amide. The minimum inhibitory concentration of spilanthol was estimated by testing different quan- tities of this compound incorporated into the PDA culture medium (potato dextrose agar). Spilanthol was incorporated into the medium at about 50°C, before it solidifi ed in

ALKALOIDS IN COSMETICS 241 the Petri dishes. The solidifi ed plates at room temperature were inoculated with a 5-mm mycelium disk, and the mycelium dry weight was determined after 10 d of incubation. S. rolfsii and S. cepivorum were more sensitive to spilanthol, with a mycelial growth inhibi- tion of 100 and 94%. A lower sensitivity was detected with Verticillium sp. and Fusarium sp. even at the higher concentration of spilanthol assayed (70). ANATABINE IN COSMETICS Anatabine (3-[(2S)-1,2, 3 ,6-tetrahydropyridin-2 - yl] pyridine) belongs to the group of pyridine alkaloids (Figure 6.). It occurs most frequently in the nightshade plants (Solan- ceae), as well as in annual paprika (Capsicum annuum L.), tomatoes (Solanum lycopersicum L.), eggplant (Solanum melongena L.), and tobacco (Nicotiana tabacum L.) (71). Because of the presence of other toxic alkaloids, e.g., nicotine, obtaining anatabine from plants is problematic. This compound is fi rst extracted with ethanol, water, steam, or carbo n diox- ide. Then, the extract is purifi ed by means of analytical techniques, e.g., liquid chroma- tography. Anatabine is alkaline in aqueous solutions, whi ch is why it is used in the production of cosmetics to neutralize the pH of the cosmetic mass. In the cosmetic indus- try, products with anatabine are most often used for the production of emulsions, creams, gels, pastes, and lotions (72,73). Anatabine has been shown to have anti-infl amma t ory effects in in vivo and in vitro studies. Studies conducted in mice, which received anatabine in dose 2 mg/kg BW, IP show that this substance reduces the level of pro-infl ammatory amyloid β (from about 4,300 pg/mL in control group to about 3,000 pg/mL in group receiving 2 mg/kg anatabine) in blood plasma (74) and inhibits the production of pro-infl ammatory cytokines such as TNFα, IL-1β, and IL-6 in tissues and blood (75). The quoted numbers are obtained by extrapolat- ing from graphs in the quoted article. The anti-infl ammatory and immunity-enhancing effects of anatabine can be used in the production of cosmetics designed to complement Figure 6. Chemical structure of anatabine (67).